An unexpected Dimroth rearrangement leading to annelated thieno [3,2-d][1,2,3]triazolo[1,5-a]pyrimidines with potent antitumor activity

Article abstract of DOI:10.1016/j.ejmech.2013.05.012

An unusual Dimroth rearrangement occurring in the reaction leading to annelated thieno[2,3-e][1,2,3] triazolo[1,5-a]pyrimidine core allowed the isolation of the linear isomer thieno[3,2-d][1,2,3]triazolo [1,5-a]pyrimidine. By decorating the linear isomer with the same chains that improved the biological activity of the angular isomers, new annelated thieno[3,2-d][1,2,3] triazolo[1,5-a]pyrimidines were designed and synthesized. They were selected by the Developmental Therapeutics Program (DTP) of the National Cancer Institute (NCI) for the anticancer screening against a panel of 60 human tumor cell lines. The biological results showed that the new derivatives exhibited strong antiproliferative activity up to nanomolar concentration. In vivo screenings of the most active compound, the N-[2-(1H-imidazol-4-yl) ethyl]-4-(3-phenyl-10-oxo- 4,10-dihydrobenzothieno[3,2-d][1,2,3]triazolo[1,5-a]pyrimidin-4-yl)butanamide, showed its low toxicity and high potency.

Full text of DOI:10.1016/j.ejmech.2013.05.012

Accepted Manuscript
An Unexpected Dimroth Rearrangement Leading to Annelated Thieno[3,2-d]
[1,2,3]triazolo[1,5-a]pyrimidines with Potent Antitumor Activity
Antonino Lauria, Chiara Patella, Ilenia Abbate, Annamaria Martorana, Anna Maria
Almerico
PII:
S0223-5234(13)00312-7
10.1016/j.ejmech.2013.05.012
EJMECH 6190
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 January 2013
Revised Date: 9 May 2013
Accepted Date: 10 May 2013
Please cite this article as: A. Lauria, C. Patella, I. Abbate, A. Martorana, A.M. Almerico, An Unexpected
Dimroth Rearrangement Leading to Annelated Thieno[3,2-d][1,2,3]triazolo[1,5-a]pyrimidines with Potent
Antitumor Activity, European Journal of Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.05.012.
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Graphical Abstract (for review)
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ACCEPTED MANUSCRIPT
Dimroth rearrangement occurred in the synthesis of annelated thieno[2,3-e][1,2,3]triazolo[1,5-
a]pyrimidine core, giving the linear isomer thieno[3,2-d][1,2,3]triazolo[1,5-a]pyrimidine.
Derivatives of this skeleton possess potent antitumor activity.

Highlights (for review)
ACCEPTED MANUSCRIPT
An Unexpected Dimroth Rearrangement Leading to
Annelated Thieno[3,2-d][1,2,3]triazolo[1,5-
a]pyrimidines with Potent Antitumor Activity
Antonino Lauria,* Chiara Patella, Ilenia Abbate, Annamaria Martorana, Anna Maria Almerico
Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche “STEBICEF”,
Sezione di Chimica Farmaceutica e Biologica - Università di Palermo, Via Archirafi 32 I-90123
Palermo, Italy.
Phone number: +390916167210; Fax number: +390916230923
Email_Address: antonino.lauria@unipa.it
Research Highlights

 An unusual Dimroth rearrangement led to new thienotriazolopyrimidine derivatives.
 The biological screenings showed their strong antiproliferative activity.
 The most active compound showed low toxicity and high potency in vivo.

Revised Manuscript
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ACCEPTED MANUSCRIPT
1
An Unexpected Dimroth Rearrangement Leading to
Annelated Thieno[3,2-d][1,2,3]triazolo[1,5-
a]pyrimidines with Potent Antitumor Activity
Antonino Lauria,* Chiara Patella, Ilenia Abbate, Annamaria Martorana, Anna Maria Almerico
Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche “STEBICEF”,
Sezione di Chimica Farmaceutica e Biologica - Università di Palermo, Via Archirafi 32 I-90123
Palermo, Italy.
Phone number: +390916167210; Fax number: +390916230923
Email_Address: antonino.lauria@unipa.it
RECEIVED DATE [to be automatically inserted after your manuscript is accepted if
required according to the journal that you are submitting your paper to]
Corresponding Author:
Prof. Antonino Lauria
Via Archirafi 32,
90123 Palermo – Italy
Tel: +39-0916167210
Fax: +39-0916230923

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ABSTRACT
An unusual Dimroth rearrangement occurring in the reaction leading to annelated thieno[2,3-
e][1,2,3]triazolo[1,5-a]pyrimidine core allowed the isolation of the linear isomer thieno[3,2-
d][1,2,3]triazolo[1,5-a]pyrimidine. By decorating the linear isomer with the same chains that
improved the biological activity of the angular isomers, new annelated thieno[3,2-
d][1,2,3]triazolo[1,5-a]pyrimidines were designed and synthesized. They were selected by the
Development Therapeutical Program (DTP) of the National Cancer Institute (NCI) for the
anticancer screening against a panel of 60 human tumor cell lines. The biological results showed
that the new derivatives exhibited strong antiproliferative activity up to nanomolar concentration.
In vivo screenings of the most active compound, the N-[2-(1H-imidazol-4-yl)ethyl]-4-(3-phenyl-
10-oxo-4,10-dihydrobenzothieno[3,2-d][1,2,3]triazolo[1,5-a]pyrimidin-4-yl)butanamide, showed
its low toxicity and high potency.
Keywords: Annelated thienotriazolopyrimidines, Domino reactions, Dimroth rearrangement,
Developmental Therapeutics Program (DTP), Anticancer agents.
Highlights:
 An unusual Dimroth rearrangement led to new thienotriazolopyrimidine derivatives.
 The biological screenings showed their strong antiproliferative activity.
 The most active compound showed low toxicity and high potency in vivo.

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1. INTRODUCTION
Heterocyclic compounds represent the most numerous class that is present among the known
drugs [1]. Typically, they are scaffolds that need to be decorated with selected substituents to
exert the biological effect. Since many years we have been interested in the design and synthesis
of compounds with antiproliferative activity. In silico design methodology (molecular docking,
chemometric protocols, etc) allowed to identify annelated pyrrolo[3,2-e]pyrimidine derivatives
of type 1 [2] and indolo[2,3-e][1,2,3]triazolo[1,5-a]pyrimidines of type 2 [3] with a wide
spectrum of antitumour activities (Figure 1).
Fig. 1.
The synthesis of annelated pyrrolo-pyrimidine core of type 1 was performed through the domino
reaction involving 2-amino-3-cyanopyrroles and N-[bis(methylthio)methylene]amino moiety
(BMMA) [4]. While in the synthesis of indolo[2,3-e][1,2,3]triazolo[1,5-a]pyrimidine core 2 was
useful employed the 1,3-cycloaddition reaction (13DCR) of ortho-azido derivatives and
acetonitriles in domino reaction [3]. The 13DCRs have been widely applied for many years in
the synthesis of polyheterocycles as scaffolds in the search of biologically active compounds [5-
9]. Moreover, the 13DCRs were recently applied in the synthesis of annelated thieno[2,3-
e][1,2,3]triazolo[1,5-a]pyrimidine cores of type 3 (Scheme 1) [10], designed through the Virtual
Lock And Key (VLAK) protocol [11].
Scheme 1.
They were synthesized starting from the ethyl 3-azido-benzo[b]thiophene-2-carboxylate (4a) or
the aza analog ethyl 3-azidothieno[2,3-b]pyridine-2-carboxylate (4b) and acetonitriles 5 (Scheme
1) to give the intermediates 6. These last were decorated through optimized protocols to give 3.

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Three of these derivatives 3aA, 3bA, and 3bB (Figure 2) showed excellent antiproliferative
activity up to sub-nanomolar concentration against a large spectrum of human tumor cell lines
[10].
Fig. 2.
2. RESULTS AND DISCUSSION
2.1 Chemistry
The first step in the synthesis of 3aA starts from the reaction of the core 7a with ethyl 4-
bromobutyrate in N,N-DiMethylFormamide (DMF) and K2CO3 (Scheme 2) [10]. In addition to
the expected product 8, the linear isomer 9 was isolated, in low yield.
Scheme 2.
This experimental evidence is not entirely surprising. In fact it is known as the pyrrolo/indolo -
[1,2,3]triazolo[1,5-a]pyrimidines 10, in basic conditions, are converted to linear isomers 11
through a Dimroth rearrangement (Scheme 3) [12,13].
Scheme 3.
Therefore, in basic conditions, the presence of the pyrimidine ring induced a partial
rearrangement to linear isomer 9, before the reaction could reach total conversion to final angular
isomer 8.
The serendipitous experimental evidence promoted us to synthesize new derivatives of the
linear isomer with the same chains that had increased the antiproliferative activity of the angular
isomer [10].
Different attempts were carried out to optimize the reaction conditions in order to address
the isolation of the linear isomer in good yields, as reported in experimental section.

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Thus, the linear isomer 9 was hydrolyzed with NaOH in EtOH/H2O mixture to give the
corresponding linear intermediate 12. Finally, the derivative 13 was obtained by the reaction of
the carboxylic acid derivative 12 with histamine and EDCI/DMAP (EDCI, 1-Ethyl-3-(3-
Dimethylaminopropyl)CarbodiImide; DMAP, 4-DiMethylAminoPyridine) (Scheme 4).
Scheme 4.
Again a partial rearrangement was observed in the reaction leading to the chloropropyl derivative
14. Thus, this reaction gave also the linear isomer 15. Upon refluxing isomer 15 in the respective
reagent under solvent-free conditions, derivative 16 was obtained (Scheme 4).
2.2 Biological screening
The new derivatives 13, 16 and the intermediates 9, 12, and 15 were subjected to the NCI
disease-oriented human cell lines screening assay to be evaluated for their in vitro antitumor
activity.
Derivatives 12, 13, and 16 passed the selection criteria adopted by DTP NCI screening and
consequently were in vitro analyzed.
A single dose (10 M) of the accepted compounds was tested against a panel of approximately
60 human tumor cell lines grouped in nine disease subpanels; namely, breast, central nervous
system, colon, leukemia, melanoma, non small-cell lung, ovarian, renal, and prostate tumors cell
lines. In table 1 the overview of one dose screening for the new derivatives 12, 13 and 16 is
reported (Supplementary material) [14-16].
Table 1.
With the aim to compare the antiproliferative activity, the biological data of the previously
angular isomers 3 were also reported [10].

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In the biological protocol, each cell line is inoculated and preincubated on a microtiter plate. Test
compounds were then added at a single concentration and the culture is incubated for 48 h. End-
point determinations were made with alamar blue [17]. Results for each tested compound are
reported as the percent of growth (G%) of the treated cells when compared to the untreated
control cells. Compounds which reduced the growth of any one of the cell lines to approximately
32% or less, are passed on for evaluation in the full panel of 60 cell lines over a 5-log dose range.
The new annelated thieno[3,2-d][1,2,3]triazolo[1,5-a]pyrimidines generally exhibited excellent
antiproliferative activity. In particular, derivatives 13 and 16 showed G% mean values of -39 and
13, respectively (Table 1).
These two derivatives exhibited G% values significantly under the level of 32% for at least one
tumor cell line. Thus, according with the selection criteria of DTP NCI protocol, they were
passed to five dose concentrations screening.
In the five dose screening, the antitumor activity is given by three parameters for each cell line:
pGI50 value (GI50 is the molar concentration of the compound that inhibits 50% of cell growth),
pTGI value (TGI is the molar concentration of the compound leading to total inhibition of cell
growth), and pLC50 value (LC50 is the molar concentration of the compound that induces 50%
cell death). Moreover, a Mean Graph MIDpoint (MG_MID) is calculated for each of the
mentioned parameters, giving an average activity parameter over all cell lines. For the
calculation of the MG_MID, insensitive cell lines are included with the highest concentration
tested. The discovery of compounds with new selectivity patterns is one of the targets of the
Developmental Therapeutical Screening Program (DTP) screening program. Selectivity of a
compound towards certain cell line is characterized by a high deviation of the particular cell line
parameter compared to the Mean Graph MIDpoint (MG_MID) value. The overview of pGI50,

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pTGI, and pLC50 values are reported in table 2, together with the number of human tumor cell
lines investigated. Also, the biological data of the previously reported angular isomers 3 [10] are
presented with the aim to compare the results.
Table 2.
Evaluation of the detailed biological data (Table 3) revealed that the derivatives selected for the
five dose screening, showed a significant antiproliferative activity against all human tumor cell
lines investigated, generally in the sub-micromolar concentration.
Table 3.
The analysis of the biological data, as MG_MID values of angular isomers 3aA, 3bA, and 3bB
[10] and the new linear isomers 13 and 16 revealed that isomer 13 is the most active compound
with pGI50 and pTGI values of 6.28 and 5.51, respectively, followed by angular isomer 3aA, and
16 (Table 3).
With respect to the tumor subpanels (Table 3), compound 13 was particularly effective against
colon cancer and leukemia cell lines. In fact, the calculated pGI50 MG_MID values for these sub-
panels (6.86 and 7.31, respectively) were always much higher than the overall MG_MID value
(6.28).
Compound 13 showed excellent activity against HCT-116 (pGI50>8.00), SF-539, (pGI50=7.69),
CCRF-CEM (pGI50=7.81), and SN12C (pGI50=7.96).
Moreover, it has to be underlined that the anticancer activity of 13 against the OVCAR-8 cell
line with pGI50>8 and pLC50<4, demonstrating the highest potency and least toxicity. A similar
behavior was observed in case of the angular isomer 3aA against OVCAR-8 (pGI50=6.49,
pLC50<4) and HCT-116 (pGI50=6.47, pLC50<4) cell lines [10].

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Particular attention should also be paid when comparing the antiproliferative activity of angular
isomers 3aA and 3bB and the corresponding linear isomers 13 and 16. Although having the same
side chain, the different structural conformation (angular or linear) of the tetracyclic system
deeply influences the biological activity.
Compound 13 with the highest pGI50 value, was selected by DTP NCI protocol for the acute
toxicity test. This consists of a rapid procedure used to measure the concentration that will make
the tested organisms sick.
Thus, three different mice received doses of 400, 200 and 100 mg/Kg of compound 13. The mice
are observed for a period of two weeks. They are sacrificed if they lose more than 20% of their
body weight or if there are other signs of significant toxicity. If all three mice sacrificed, then the
next three dose levels (50, 25, 12.5 mg/kg) should be tested in a similar way. This process is
repeated until a tolerated dose is found.
The obtained data showed that compound 13 was not toxic at all tested concentrations; no
negative effects were observed in the screened mice for the two weeks of tests.
Therefore, compound 13 is considered to be a good candidate as anticancer agent.
Further in vivo analyses and studies are in progress by DTP NCI, to elucidate the mechanism of
action, and will be reported elsewhere.
The biological data provided by DTP NCI screening were useful to analyze the different
biological activity spectrum of the angular and linear isomers. Since many years, the analysis
COMPARE [18] is available from DTP NCI. This facility is able to match the biological data of
a screened compound with specific databases, with the aim of finding the drug that best fits the
input compound.

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The most active compound 13 tested as seed against the NCI “Standard Agents” database,
showed a Pearson correlation coefficient (PCC) of 0.551 for Aclacinomycin A and of 0.470 for
N,N-dibenzyldaunomycin, known inhibitors of topoisomerase II.
Recently, CellMiner, an evolution of COMPARE analysis, was developed [19]. This is a web-
based suite of Genomic and Pharmacologic Tools to explore transcript and drug patterns in the
NCI-60 cell line set. Thus, by submitting the NCI DTP results of the most active compound 13
and its angular isomer 3aA, it emerged that they have different biological profiles. In fact, the
PCC of 13 on 3aA is 0.716 and the significant gene correlations and the microRNA correlations
showed a quite different patterns (supplementary material).
3. CONCLUSIONS
An unusual Dimroth rearrangement in the reaction leading to annelated thieno[2,3-
e][1,2,3]triazolo[1,5-a]pyrimidine core allowed to identify annelated thienotriazolopyrimidines
with antitumor activity up to nanomolar concentration. The derivative N-[2-(1H-Imidazol-4-
yl)ethyl]-4-(3-phenyl-10-oxo-4,10-dihydrobenzothieno[3,2-d][1,2,3]triazolo[1,5-a]pyrimidin-4-
yl)butanamide 13 revealed to be an excellent candidate as antitumor agent. In fact, it showed
antiproliferative activity against all tested tumor cell lines, up to nanomolar concentration.
Moreover, preliminary in vivo tests showed that it has high potency and low cytotoxicity. Two
factors contributed to the identification of this compound: the computational approach VLAK
applied taking the advantage of old biological results on indolo[2,3-e][1,2,3]triazolo[1,5-
a]pyrimidines, and the serendipitous synthesis that drove to build the linear core of type 13.
Finally, this scaffold represents a new ring system that can be used as lead in further
optimization. The statistical analysis of the biological data of the designed compounds,

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performed through COMPARE and CellMiner Tools, allowed to underline the difference in
biological property between the angular and linear isomers.
4. EXPERIMENTAL
4.1. Chemistry
Unless otherwise indicated, all reagents and solvents were purchased from commercial sources
and used without further purification. All melting points (°C) were determined on a Büchi–
Tottoli capillary apparatus and are uncorrected; IR spectra were determined in bromoform with a
Jasco FT/IR 5300 spectrophotometer. 1H NMR, 13C NMR spectra were recorded in DMSO-d6
solution, unless otherwise specified, at 200 and 50.3 MHz, respectively, using a Bruker AC-E
series 200 MHz spectrometer. Chemical shift values are given in ppm and referred as the internal
standard to TMS (tetramethylsilane). The purity of all compounds screened in biological assays
was determined to be >95% by HPLC/MS analysis. Mass spectroscopy was performed using a
GC-MS Shimadzu QP5050 with EI (75ev). Microanalyses were in agreement with theoretical
values ±0.4%. Thin layer chromatography was performed on precoated (0.25 mm) silica gel
GF254 plates, compounds were detected with 254nm UV lamp. Column chromatography was
performed with Merck silica gel ASTM (230–400 mesh), or Merck aluminium oxide 90 (70-230
mesh), and with a Biotage FLASH40i chromatography module (prepacked cartridge system).
4.1.1. General procedure for preparation of ethyl 4-(5-oxo-3-phenylbenzo[4,5]thieno[2,3-
e][1,2,3]triazolo[1,5-a]pyrimidin-4(5H)-yl)butanoate (8) and ethyl 4-(10-oxo-3-
phenylbenzo[4,5]thieno[3,2-d][1,2,3]triazolo[1,5-a]pyrimidin-4(10H)-yl)butanoate (9).
To a stirred suspension of 7a (1.0 mmol) in N,N-dimethylformamide (5 mL), potassium
carbonate (3.0 mmol) and ethyl 4-bromobutyrate (5.0 mmol) were added. The mixture was

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refluxed for 4 h, cooled to room temperature, and then slowly poured onto ice-water. The crude
solid was filtered and purified by column chromatography using dichloromethane/ethyl acetate
98:2 as eluent to afford both 8 (0.11 g, yield 26%), and 9 (0.30 g. yield 70%).
4.1.1.1. Ethyl 4-(5-oxo-3-phenylbenzo[4,5]thieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidin-4(5H)-
yl)butanoate (8): Mp 123.6-124.0°C. IR: 1725, 1677 (2CO) cm-1. 1H NMR (CDCl3) δ: 1.22 (t, J=
7.4 Hz, 3H, OCH2CH3), 2.07-2.15 (m, 2H, CH2CH2CH2), 2.41 (t, J= 7.2 Hz, 2H, CH2CO), 4.04-
4.12 (q, J= 7.4 Hz, 2H, OCH2CH3), 5.04 (t, J= 7.2 Hz, 2H, NCH2), 7.49-7.62 (m, 5H, C8-H, C9-
H, C3’-H, C4’-H, C5’-H), 7.90 (d, J= 7.4 Hz, 1H, C7-H), 8.48 (d, J= 7.4 Hz, 1H, C10-H), 8.60
(d, J= 8.0 Hz, 2H, C2’-H, C6’-H). 13C NMR (CDCl3) δ: 14.1 (q), 23.6 (t), 31.1 (t), 55.3 (t), 60.7
(t), 115.7 (s), 123.4 (d), 124.3 (d), 125.0 (d), 127.0 (dx2), 128.6 (s), 128.8 (dx2), 129.5 (d), 129.9
(d), 134.1 (s), 139.2 (s), 142.1 (s), 145.9 (s), 152.0 (s), 155.5 (s), 172.2 (s). Elem. Anal. calcd. for
C23H20N4O3S: C, 63.87; H, 4.66; N, 12.95; found: C, 63.61; H, 4.83; N, 12.54.
4.1.1.2.
Ethyl
4-(10-oxo-3-phenylbenzo[4,5]thieno[3,2-d][1,2,3]triazolo[1,5-a]pyrimidin-
1
4(10H)-yl)butanoate (9): Mp 217.2-217.7°C. IR: 1730, 1690 (2CO) cm-1. H NMR (CDCl3) δ:
1.26 (t, J= 7.4 Hz, 3H, OCH2CH3), 2.21-2.34 (m, 2H, CH2CH2CH2), 2.59 (t, J= 7.3 Hz, 2H,
CH2CO), 4.12-4.20 (q, J= 7.4 Hz, 2H, OCH2CH3), 4.67 (t, J= 7.3 Hz, 2H, NCH2), 7.24-7.63 (m,
5H, C6-H, C7-H, C3’-H, C4’-H, C5’-H), 7.90 (d, J= 7.0 Hz, 1H, C8-H), 8.35 (d, J= 8.1 Hz, 2H,
13
C2’-H, C6’-H), 9.04 (d, J= 7.0 Hz, 1H, C5-H). C NMR (CDCl3) δ: 14.2 (q), 24.0 (t), 30.9 (t),
60.7 (t), 67.2 (t), 123.1 (d), 125.6 (d), 126.2 (d), 126.5 (dx2), 127.4 (d), 128.0 (s), 128.5 (s),
128.6 (dx2), 129.5 (d), 131.1 (s), 133.2 (s), 135.2 (s), 137.9 (s), 140.4 (s), 157.6 (s), 172.9 (s).
Elem. Anal. calcd. for C23H20N4O3S: C, 63.87; H, 4.66; N, 12.95; found: C, 63.93; H, 4.74; N,
12.87.

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4.1.2.
4-(10-Oxo-3-phenylbenzo[4,5]thieno[3,2-d][1,2,3]triazolo[1,5-a]pyrimidin-4(10H)-
yl)butanoic acid (12).
To a stirred suspension of the ethoxycarbonyl derivative 9 (0.5 mmol) in absolute ethanol (5
mL), a solution of potassium hydroxide (0.13 g, 2.5 mmol) in water (2 mL) was added dropwise.
The mixture was stirred at room temperature for 1-8 h. The solvent was evaporated under
reduced pressure. The residue was dissolved in water (10 mL) and then carefully adjusted to
pH=1 with 6N hydrochloric acid. The precipitated solid was filtered, dried and recrystallized
from ethanol to afford the corresponding acid 12 as white solid, 0.40 g. Yield 100%. Mp 219.3-
1
220.0°C. IR: 3519-2900 (OH), 1720, 1681 (2CO) cm-1. H NMR δ: 1.94-2.05 (m, 2H,
CH2CH2CH2), 2.33 (t, J= 7.4 Hz, 2H, CH2CO), 4.96 (t, J= 7.4 Hz, 2H, NCH2), 7.52-7.69 (m, 5H,
C6-H, C7-H, C3’-H, C4’-H, C5’-H), 8.12 (d, J= 7.3 Hz, 1H, C8-H), 8.38 (d, J= 7.3 Hz, 1H, C5-
H), 8.52 (d, J= 8.0 Hz, 2H, C2’-H, C6’-H), 10.62 (s, 1H, COOH). 13C NMR δ: 22.8 (t), 29.8 (t),
54.5 (t), 123.0 (d), 123.3 (d), 124.0 (s), 124.7 (d), 125.8 (dx2), 128.0 (s), 128.1 (d), 128.2 (dx2),
129.1 (d), 131.2 (s), 133.4 (s), 135.4 (s), 137.9 (s), 140.7 (s), 157.5 (s), 173.9 (s). Elem. Anal.
calcd. for C21H16N4O3S: C, 62.36; H, 3.99; N, 13.85; found: C, 62.43; H, 3.87; N, 13.89.
4.1.3.
N-(2-(1H-Imidazol-4-yl)ethyl)-4-(10-oxo-3-phenylbenzo[4,5]thieno[3,2-
d][1,2,3]triazolo[1,5-a]pyrimidin-4(10H)-yl)butanamide (13).
To a stirred suspension of the acid 12 (0.44 mmol) in dioxane (5 mL), 4-dimethylaminopyridine
(DMAP), 0.05 g, 0.45 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI)
(0.17g, 9.2 mmol) were added under nitrogen atmosphere, at 0°C. After 2 h of stirring at room
temperature, histamine (0.102 g, 9.2 mmol) was carefully added and the reaction mixture was
incubated at 50°C for 12h. The reaction mixture was evaporated under reduced pressure and the
crude product was purified by column chromatography using dichloromethane/methanol 9:1 as

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eluent. Pale yellow solid, 0.15 g. Yield 70%. Mp 140.7-141.2°C. IR: 3360 (NH), 1722, 1650
1
(2CO) cm-1. H NMR δ: 1.96-2.05 (m, 2H, CH2CH2CH2), 2.17 (t, J= 7.4 Hz, 2H, COCH2), 2.53
(t, J= 7.3 Hz, 2H, CH2CH2NH), 3.17 (t, J= 7.3 Hz, 2H, CH2CH2NH), 4.97 (t, J= 7.4 Hz, 2H,
NCH2), 6.72 (s, 1H, C5”-H), 7.41-7.99 (m, 8H, C6-H, C7-H, C3’-H, C4’-H, C5’-H, C2”-H,
2NH), 8.16 (d, J= 7.4 Hz, 1H, C8-H), 8.44 (d, J= 7.4 Hz, 1H, C5-H), 8.56 (d, J= 8.1 Hz, 2H, C2’-
13
H, C6’-H). C NMR δ: 24.1 (t), 26.9 (t), 32.0 (t), 40.7 (t), 55.5 (t), 122.2 (d), 123.7 (s), 124.1
(d), 124.9 (d), 125.5 (d), 126.5 (dx2), 128.4 (d), 128.9 (dx2), 129.7 (d), 131.1 (s), 133.7 (d),
134.6 (s), 135.5 (s), 137.3 (s), 141.1 (s), 142.3 (s), 151.4 (s), 154.5 (s), 170.7 (s). Elem. Anal.
calcd. for C26H23N7O2S: C, 62.76; H, 4.66; N, 19.71; found: C, 62.83; H, 4.72; N, 19.67.
4.1.4.
General
procedure
for
preparation
of
4-(3-chloropropyl)-3-
phenylpyrido[3',2':4,5]thieno[3,2-d][1,2,3]triazolo[1,5-a]pyrimidin-10(4H)-one (15).
To a stirred suspension of 7b (0.62 g, 1.93 mmol) in N,N-dimethylformamide (12 mL),
potassium carbonate (0.8 g, 5.81 mmol) and 1-bromo-3-chloropropane (1.52 g, 5,79 mmol) were
added. The mixture was refluxed for 5 h, cooled to room temperature, and then slowly poured
onto ice-water. The precipitate solid was filtered and purified by column chromatography using
dichloromethane/ethyl acetate 98:2 as eluent to afford both 14 (0.12 g, yield 15%), and 15 (0.51
g. yield 67%).
4.1.4.1. 4-(3-Chloropropyl)-3-phenylpyrido[3',2':4,5]thieno[2,3-e][1,2,3]triazolo[1,5-
a]pyrimidin-5(4H)-one (14). Pale yellow solid. Mp 200.8-201.7°C. IR: 1645 (CO) cm-1. 1H
NMR (CDCl3) δ: 2.28-2.43 (m, 2H, CH2CH2CH2), 3.63 (t, J= 6.3 Hz, 2H, CH2Cl), 5.13 (t, J= 6.3
Hz, 2H, CH2N), 7.47-7.57 (m, 4H, C9-H, C3’-H, C4’-H, C5’-H), 8.56 (d, J= 8.2 Hz, 2H, C2’-H,
C6’-H), 8.70 (dd, J=1.7, 6.6 Hz, 1H, C10-H), 8.79 (dd, J=1.7, 4.6 Hz, 1H, C8-H). 13C NMR δ:
31.2 (t), 41.3 (t), 53.6 (t), 100.0 (s), 115.0 (s), 120.2 (d), 127.0 (dx2), 128.2 (s), 128.8 (dx2),

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14
130.2 (d), 132.2 (d), 139.2 (s), 145.7 (s), 151.5 (d), 151.9 (s), 152.9 (s), 163.4 (s). Elem. Anal.
calcd. for C19H14ClN5OS: C, 57.65; H, 3.56; N, 17.69; found: C, 57.76; H, 3.39; N, 17.43.
4.1.4.2.
4-(3-Chloropropyl)-3-phenylpyrido[3',2':4,5]thieno[3,2-d][1,2,3]triazolo[1,5-
1
a]pyrimidin-10(4H)-one (15). White solid. Mp 195.4-196.6°C. IR: 1630 (CO) cm-1. H NMR
(CDCl3) δ: 2.45-2.59 (m, 2H, CH2CH2CH2), 3.84 (t, J= 6.2 Hz, 2H, CH2Cl), 4.93 (t, J= 6.2 Hz,
2H, CH2N), 7.33-7.41 (m, 3H, C3’-H, C4’-H, C5’-H), 7.54 (dd, J= 4.6, 7.2 Hz, 1H, C6-H), 7.70
(d, J= 8.2 Hz, 2H, C2’-H, C6’-H), 8.92 (dd, J= 1.7, 4.6 Hz, 1H, C5-H), 9.40 (dd, J= 1.7, 7.2 Hz,
1H, C7-H). 13C NMR (CDCl3) δ: 29.7 (t), 41.1 (t), 65.1 (t), 121.3 (d), 122.5 (d), 125.8 (dx2),
127.7 (d), 128.7 (dx2), 130.7 (s), 133.1 (s), 134.0 (s), 134.2 (s), 134.4 (d), 137.7 (s), 151.6 (s),
157.4 (s), 161.7 (s). Elem. Anal. calcd. for C19H14ClN5OS: C, 57.65; H, 3.56; N, 17.69; found: C,
57.59; H, 3.64; N, 17.63.
4.1.5. General procedure for preparation of
4-(3-(4-methylpiperazin-1-yl)propyl)-3-
phenylpyrido[3',2':4,5]thieno[3,2-d][1,2,3]triazolo[1,5-a]pyrimidin-10(4H)-one (16).
A stirred suspension of 15 (0.10 g, 0.25 mmol) in 1-methylpiperazine (3 mL) was heated under
reflux for 2 h. After cooling the mixture was poured onto ice-water and the precipitate solid was
filtered. Purified by alumina column chromatography using dichloromethane as eluent, to afford
1
16 as a yellow solid, 0.05 g. Yield 40%. Mp 149.6-150.8°C. IR: 1635 (CO) cm-1. H NMR
(CDCl3) δ: 2.14-2.24 (m, 2H, CH2CH2CH2), 2.29 (s, 3H, CH3), 2.49-2.67 (m, 10H,
CH2N(CH2CH2)2N), 4.77 (t, J= 6.6 Hz, 2H, CH2N), 7.28-7.51 (m, 3H, C3’-H, C4’-H, C5’-H),
7.65 (dd, J= 4.6, 7.4 Hz, 1H, C6-H), 8.39 (d, J= 8.0 Hz, 2H, C2’-H, C6’-H), 8.88 (dd, J= 1.7, 4.6
Hz, 1H, C5-H), 9.31 (dd, J= 1.7, 7.4 Hz, 1H, C7-H). 13C NMR (CDCl3) δ: 26.1 (t), 46.0 (q), 53.3
(t), 55.0 (tx2), 55.1 (tx2), 67.0 (t); 112.9 (s), 121.2 (d), 122.4 (s), 125.7 (dx2), 127.6 (d), 128.6

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15
(dx2), 130.8 (s), 132.8 (s), 133.6 (s), 134.3 (d), 137.7 (s), 151.4 (d), 157.6 (s), 161.6 (s). Elem.
Anal. calcd. for C24H25N7OS: C, 62.72; H, 5.48; N, 21.33; found: C, 62.82; H, 5.44; N, 21.37.
4.2. Biology
The human tumor cell lines of the cancer screening panel are grown in RPMI 1640 medium
containing 5% fetal bovine serum and 2 mM L-glutamine. For a typical screening experiment,
cells are inoculated into 96 multiwell microtiter plates in 100 µl at plating densities ranging from
5.000 to 40.000 cells/well depending on the doubling time of individual cell lines. After cell
inoculation, the microtiter plates are incubated at 37°C, 5 % CO2, 95 % air and 100 % relative
humidity for 24 h prior to addition of tested compounds.
After 24 h, two plates of each cell line are fixed in situ with trichloro acetic acid (TCA), to
represent a measurement of the cell population for each cell line at the time of drug addition
(time zero, Tz). Tested compounds are solubilized in dimethyl sulfoxide at 400-fold the desired
final maximum test concentration and stored frozen prior to use. At the time of compound
addition, an aliquot of frozen concentrate is thawed and diluted to twice the desired final
maximum test concentration with complete medium containing 50 µg/mL gentamicin.
Additional four, ten-fold or ½ log serial dilutions are made to provide five concentrations of the
test compounds plus control. Aliquots of 100 µl of the different drug dilutions are added to the
appropriate microtiter wells already containing 100 µl of medium, resulting in the required final
concentrations.
Following drug addition, the plates are incubated for an additional 48 h at 37°C, 5 % CO2, 95 %
air, and 100 % relative humidity. For adherent cells, the assay is terminated by the addition of
cold TCA. Cells are fixed in situ by the gentle addition of 50 µl of cold 50 % (w/v) TCA (final
concentration, 10 % TCA) and incubated for 60 min at 4°C. The supernatant is discarded, and

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16
the plates are washed five times with tap water and air dried. Sulforhodamine B (SRB) solution
(100 µl) at 0.4 % (w/v) in 1 % acetic acid is added to each well, and plates are incubated for 10
min at room temperature. After staining, unbound dye is removed by washing five times with 1
% acetic acid and the plates are air-dried. Bound stain is subsequently solubilized with 10 mM
trizma base, and the absorbance is read on an automated plate reader at a wavelength of 515 nm.
For suspension cells, the methodology is the same except that the assay is terminated by fixing
settled cells at the bottom of the wells by gently adding 50 µl of 80 % TCA (final concentration,
16 % TCA). Using the seven absorbance measurements [time zero, (Tz), control growth, (C),
and test growth in the presence of drug at the five concentration levels (Ti)], the percentage
growth is calculated at each of the drug concentrations levels. Percentage growth inhibition is
calculated as:
[(Ti-Tz)/(C-Tz)] x 100 for concentrations for which Ti>/=Tz
[(Ti-Tz)/Tz] x 100 for concentrations for which Ti<Tz.
Three dose response parameters are calculated for each tested compounds. Growth inhibition of
50 % (GI50) is calculated from [(Ti-Tz)/(C-Tz)] x 100 = 50, which is the drug concentration
resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control
cells during the drug incubation. The drug concentration resulting in total growth inhibition
(TGI) is calculated from Ti = Tz. The LC50 (concentration of drug resulting in a 50% reduction
in the measured protein at the end of the drug treatment as compared to that at the beginning)
indicating a net loss of cells following treatment is calculated from [(Ti-Tz)/Tz] x 100 = -50.
Values are calculated for each of these three parameters if the level of activity is reached;
however, if the effect is not reached or is exceeded, the value for that parameter is expressed as
greater or less than the maximum or minimum concentration tested.

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17
5. SUPPLEMENTARY MATERIAL
SM1: 1H and 13C NMR spectra for derivatives 13 and 16; DTP NCI biological data for all
compounds: one dose screening (derivatives 12, 13, 16), five dose screening (derivatives 13, 16),
and in vivo toxicity report (derivative 13). SM2: Compare and CellMiner analyses.
ACKNOWLEDGMENTS
Thanks are due to the NCI, Bethesda, MD (USA) for performing the antitumor testing of the
synthesized compounds.
REFERENCES
[1] R. Dua, S. Shrivastava, S. K.Sonwane, S. K. Srivastava, Pharmacological significance of
synthetic heterocycles scaffold: a review, Adv. Biol. Res. 5 (2011) 120-144.
[2] A. Lauria, C. Patella, I. Abbate, A. Martorana, A.M. Almerico, Lead optimization through
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[8] A. Lauria, A. Guarcello, G. Macaluso, G. Dattolo, A.M. Almerico, Reactivity of asymmetric
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Thieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidines, with Potent Anticancer Activity, Designed
through VLAK Protocol, Eur. J. Med. Chem. 62 (2013) 416-424.
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ACCEPTED MANUSCRIPT
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Transcript and Drug Patterns in the NCI-60 Cell Line Set, Cancer Res. 72 (2012) 3499-3511.

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LIST OF CAPTIONS:
Figure 1. Annelated pyrrolo[3,2-e]pyrimidine derivatives 1 and indolo[2,3-e][1,2,3]triazolo[1,5-
a]pyrimidine derivatives 2 with antitumor activities.
Figure 2. Annelated thieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidines with good antitumor activity
(3aA, pGI50 = 4.73 - 6.74; 3bA, pGI50 = 4.47 - 7.02; 3bB, pGI50 = 5.03 - 6.80).
Scheme 1. Synthetic route to annelated thieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidine derivatives.
Scheme 2. Synthesis of ethyl 4-(5-oxo-3-phenylbenzo[4,5]thieno[2,3-e][1,2,3]triazolo[1,5-
a]pyrimidin-4(5H)-yl)butanoate (8) and its linear isomer 9.
Scheme 3. Dimroth rearrangement of annelated [1,2,3]triazolo[1,5-a]pyrimidines.
Scheme 4. Synthesis of linear isomers of the targeted compounds.
Table 1. Overview of one dose screening for compounds 3aA, 3bA, 3bB, 12, 13 and 16 against
all sub-panels (G%).
Table 2. Overview of five dose screening tests results for compounds 13, 16 and the angular
isomers 3.
Table 3. DTP NCI five dose screening for compounds 13, 16 and the angular isomers 3.

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Figure 1. Annelated pyrrolo[3,2-e]pyrimidine derivatives 1 and indolo[2,3-e][1,2,3]triazolo[1,5-
a]pyrimidine derivatives 2 with antitumor activities.
Figure 2. Annelated thieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidines with good antitumor activity
(3aA, pGI50 = 4.73 - 6.74; 3bA, pGI50 = 4.47 - 7.02; 3bB, pGI50 = 5.03 - 6.80).
Scheme 1. Synthetic route to annelated thieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidine derivatives.

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Scheme 2. Synthesis of ethyl 4-(5-oxo-3-phenylbenzo[4,5]thieno[2,3-e][1,2,3]triazolo[1,5-
a]pyrimidin-4(5H)-yl)butanoate (8) and its linear isomer 9.
Scheme 3. Dimroth rearrangement of annelated [1,2,3]triazolo[1,5-a]pyrimidines.

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23
Scheme 4. Synthesis of linear isomers of the targeted compounds.

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24
Table 1. Overview of one dose screening for compounds 3aA, 3bA, 3bB, 12, 13 and 16 against
all sub-panels (G%).
Derivative (#G%)
PANEL
BreastCancer
CNS Cancer
Colon Cancer
Leukemia
3aA 3bA 3bB
-5
12
13
-28
-50
-55
55
-70
-32
-41
-92
-51
16
26
15
-13
-1
48
-6
-20
-11
47
79
60
49
61
71
88
59
46
79
-23 79
-43 88
-37 90
-22 102
-78 95
14 85
-18
-49
71
-57
-26
-16
-41
-34
-21
Melanoma
Non-Small Cell Lung Cancer
OvarianCancer
Prostate Cancer
RenalCancer
7
105
39 92
-14 75
90
*Overall average
69
-23
-39
13
#Subpanel cellular growth percentage average. *In bold the G% mean of compounds passed on for evaluation in
the full panel of 60 cell lines over a five-log dose range.
Table 2. Overview of five dose screening tests results for compounds 13, 16 and the angular
isomers 3.
Compd
pGI50
range
pTGI
range
52 <4.00-5.78
59 <4.00-5.35
pLC50
range
44 <4.00-5.35
59 <4.00-4.41
4.39-5.42
49 <4.00-5.50
49 <4.00-5.24
#
MG_MID
5.81
#
MG_MID
5.29
#
MG_MID
4.62
54 4.73-6.74
59 4.47-7.02
59 5.03-6.80
56 5.29->8.00
57 4.70-5.99
3aA
3bA
3bB
13
5.29
5.30
6.28
5.59
4.58
4.94
5.51
5.26
4.15
4.60
4.89
4.78
59
54 <4.00-6.91
57 4.38-5.61
4.75-5.76
59
16
# Number of cell lines investigated

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25
Table 3. DTP NCI five dose screening for compounds 13, 16 and the angular isomers 3.
Derivative
3aA
3bA
3bB
13
16
#CELL
PANEL
LINE
BT-549
HS 578T
MCF7
4.76 4.38 <4.00
NT
NT
NT
NT
NT
NT
5.48 4.94 4.29 4.83 4.51 4.19
NT 4.83 4.38 4.00
4.75 4.00 4.00 5.11 4.82 4.53 5.94 5.58 5.21 5.81 5.52 5.24
5.76 5.39
5.84 5.51
NT
5.19
4.95 4.50 4.06 5.19 4.79 4.40 5.83 5.42
MDA-MB-
231/ATCC
MDA-MB-
468
5.72 5.44
5.77 5.43
5.17
4.75 4.46 4.17 5.16 4.87 4.57 5.73 5.46 5.18 5.77 5.45
NT
5.10
5.09 4.56 4.10 5.16 4.86 4.55 6.47 5.92 5.42 5.74 5.48 5.22
T-47D
5.85 5.35 <4.00 5.73 4.67 4.19 5.10 4.80 4.50 5.90 5.20 4.51 4.70 4.42 4.15
panel
average 5.62 5.25
4.69
5.11
NT
5.05 4.44 4.10 5.14 4.83 4.51 5.89 5.42 4.92 5.28 4.96 4.56
4.93 4.54 4.14 5.19 4.88 4.57 5.81 5.49 5.17 5.79 5.48 5.17
5.76 4.79 4.27 5.25 4.91 4.57 6.34 5.68 5.27 5.71 5.43 5.16
6.50 4.83 4.27 5.24 4.87 4.51 7.69 6.91 5.50 5.77 5.48 5.18
SF-268
5.75 5.43
5.77 5.44
6.18 5.66
SF-295
SF-539
SNB-19
SNB-75
U251
5.24
5.77 5.30 <4.00 4.97 4.47 4.00 5.08 4.80 4.53 5.29 4.66 4.27 5.60 5.25 4.00
5.73 5.39
5.98 5.65
5.06
5.33
4.82 4.54 4.26 5.17 4.88 4.59 5.71 5.45 5.20 5.11 4.66 4.31
5.89 4.83 4.34 5.28 4.95 4.62 6.78 5.73 5.37 5.74 5.46 NT
panel
average 5.86 5.48
4.95
5.48 4.67 4.21 5.20 4.88 4.57 6.27 5.65 5.13 5.62 5.29 4.76
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
5.96 5.32 <4.00 5.20 4.63 4.23 5.13 4.84 4.54 6.48 5.65 5.23 5.79 5.49 5.19
5.75 5.38
5.01
5.69 4.52 4.00 5.17 4.87 4.58
NT
NT
NT
NT
NT
5.73 5.46 5.19
5.77 5.46 5.16
6.47 5.78 <4.00 6.42 5.00 4.41 5.28 4.93 4.58 8.00
5.90 5.60
5.87 5.50
5.94 5.55
5.30
5.13
NT
5.24 4.00 4.00 5.21 4.88 4.55 6.43 5.72 5.24 5.74 5.41 5.08
5.41 4.26 4.00 5.54 5.02 4.63 6.17 5.70 5.34 NT NT NT
5.72 4.62 4.08 5.37 4.97 4.61 6.47 5.70 5.31 5.76 5.47 5.17
6.48 4.84 4.21 5.27 4.94 4.61 7.62 6.00 5.38 5.77 5.43 5.09
KM12
SW-620
6.39
NT
NT
panel
average 6.04 5.52
4.69
5.74 4.55 4.13 5.28 4.92 4.59 6.86 5.75 5.30 5.76 5.45 5.15
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
NT
4.00 <4.00 6.65 5.35 4.00 5.81 5.28 4.61 7.81 6.54 5.28 5.78 5.37 4.00
5.85 5.20 <4.00 5.85 5.32 4.30 6.10 5.76 5.42 7.14 5.49 4.64 5.73 5.39 5.04
6.36
5.93
NT
NT
NT
NT
NT
NT
NT
6.51 4.81 4.00 6.06 5.69 5.32 7.64 5.99 5.29 5.72 5.32 4.00
5.65 4.62 4.00 5.81 5.31 4.67
NT
NT
NT
NT
NT
NT
5.70 5.35 4.94
5.74 5.36 4.00
<4.00 5.29 4.45 4.00 5.21 4.80 4.39
NT
NT
5.60 5.21 4.00 5.96 5.59 4.92 6.66 6.18 5.30 5.65 5.32
NT
panel
average 6.05 4.60 <4.00 5.93 4.96 4.05 5.83 5.41 4.89 7.31 6.05 5.13 5.72 5.35 4.40
LOX IMVI
M14
5.80 5.53
5.77 5.42
5.27
NT
4.91 4.58 4.24 5.27 4.94 4.60 5.82 5.54 5.26 5.78 5.47
5.46 4.71 4.30 5.67 5.13 4.68 6.49 5.69 NT 5.73 5.44 5.14
NT
MALME-
3M
5.60 5.26 <4.00 4.74 4.43 4.12 5.03 4.75 4.47 5.80 5.48 5.17 5.71 5.44 5.17
MDA-MB-
435
SK-MEL-2
5.83 5.51
NT NT
5.20
NT
6.31 4.69 4.00 5.93 5.52 4.88 6.46 5.73 5.33 5.73 5.45 5.17
4.75 4.41 4.07 5.06 4.78 4.50 NT NT NT NT NT NT
SK-MEL-28 5.76 5.49
5.23
5.05
4.77 4.46 4.14 5.06 4.80 4.55 5.77 5.50 5.23 4.80 4.53 4.26
4.79 4.00 4.00 5.14 4.86 4.58 5.69 4.96 4.48 5.77 5.50 5.24
SK-MEL-5
UACC-257
UACC-62
5.72 5.39
5.69 5.34 <4.00 4.74 4.45 4.16 5.64 5.12 4.69 5.67 5.39 5.11 5.74 5.47 5.20
5.72 5.45
5.18
4.80 4.52 4.24 5.05 4.79 4.53 5.74 5.45 5.15 4.78 4.46 4.15
panel
average 5.74 5.42
A549/ATCC 5.68 5.34
4.85
NT
5.03 4.47 4.14 5.32 4.97 4.61 5.93 5.47 5.10 5.51 5.22 4.90
5.06 4.46 4.00 5.17 4.86 4.55 5.89 5.49
NT
5.75 5.47
NT
EKVX
5.57 5.06 <4.00 4.82 4.35 4.00 5.19 4.88 4.56 5.60 4.80 4.00 5.67 5.31 4.08
5.64 5.28 <4.00 4.78 4.50 4.21 5.09 4.81 4.54 5.88 5.54 5.20 5.75 5.48 5.21
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M 5.65 5.26
NCI-H460
NCI-H522
5.73
4.73 4.42
5.53 4.93 <4.00 4.84 4.49 4.15 5.09 4.81 4.54 5.87 5.45
NT
<4.00 4.77 4.40 4.04 5.24 4.89 4.54 5.39 4.69 4.00 5.20 4.61 4.15
4.12 4.93 4.53 4.13 5.13 4.82 4.50 5.56 5.13 4.39 4.86 4.53 4.19
NT 5.67 5.29 4.47
4.83 4.44 4.05 5.21 4.89 4.57 5.56 5.01 4.41 5.76 5.46 5.16
5.44 4.88 4.37 5.16 4.86 4.57 5.80 5.40 NT 5.72 5.40 5.09
5.53 4.64 4.22 5.14 4.84 4.54 7.36 5.51 4.00 5.72 5.43 NT
5.00 4.52 4.13 5.16 4.85 4.55 5.88 5.22 4.33 5.57 5.22 4.72
4.67
5.11
4.09
4.25
5.73 5.42
5.98 4.93
panel 5.58 5.08

ACCEPTED MANUSCRIPT
26
average
IGROV1
NT
NT
NT
5.02 4.56 4.12 5.23 4.86 4.49 7.00 5.68 5.30 5.70 5.35 5.01
NCI/ADR-
RES
5.49 4.00 <4.00 4.47 4.00 4.00 5.14 4.85 4.55 5.97 4.00 4.00 5.65 5.24 4.36
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
5.82 5.50
5.76 5.42
5.92 5.56
5.18
NT
NT
4.96 4.63 4.30 5.64 5.12 4.69 5.78 5.51 5.25 5.74 5.46 5.18
4.81 4.49 4.17 5.14 4.85 4.57 5.74 5.42 5.10 5.73 5.41 5.09
4.94 4.49 4.03 5.05 4.77 4.49 7.25 5.87 5.39 5.57 5.13 4.00
6.49 5.55 <4.00 7.02 4.76 4.17 5.11 4.81 4.52 8.00 5.52 4.00 5.71 5.35 4.00
5.46 4.81
4.18
4.84 4.56 4.27 5.05 4.80 4.55 5.62 5.07 4.53 4.73 4.48 4.22
panel
average 5.82 5.14
DU-145 5.81 5.53
PC-3 NT
4.34
5.24
5.15 4.50 4.15 5.19 4.87 4.55 6.48 5.30 4.80 5.55 5.20 4.55
5.56 4.71 4.21 5.23 4.91 4.60 5.97 5.65 5.32 5.73 5.44 5.14
4.00 <4.00 4.96 4.00 4.00 5.20 4.87 4.54 7.22 5.66 5.14
NT
NT
NT
panel
average 5.81 4.77
4.62
NT
5.24
5.26
5.35
5.26 4.36 4.11 5.22 4.89 4.57 6.60 5.66 5.23 5.73 5.44 5.14
786-0
A498
5.80 5.45
5.81 5.52
5.78 5.52
6.74 5.77
4.90 4.59 4.28 5.13 4.84 4.54 5.98 5.63 5.27 5.73 5.46 5.18
5.17 4.65 4.27 6.80 5.03 4.66 5.96 5.61 5.25 4.95 4.62 4.28
4.81 4.51 4.20 5.09 4.82 4.56 5.80 5.50 5.20 5.79 5.48 5.17
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
5.54 4.84 4.14 5.21 4.89 4.56
NT
NT
NT
5.79 5.48 5.18
5.60 5.30 <4.00 4.83 4.47 4.11 5.20 4.87 4.55 5.66 5.38 5.11 5.77 5.50 5.23
6.57 5.73
5.67 5.40
5.72 5.43
5.29
NT
NT
6.48 4.87 4.38 5.14 4.85 4.55 7.96 5.69 5.26 5.74 5.39
4.87 4.55 4.24 5.16 4.87 4.57 5.66 5.43 5.19 5.67 5.43
NT
NT
4.93 4.57 4.22 5.29 4.92 4.55 5.89 5.58 5.28 5.99 5.61 5.23
panel
average 5.96 5.52
MG_MID 5.81 5.29
5.03
4.62
5.19 4.63 4.23 5.38 4.89 4.57 6.13 5.55 5.22 5.68 5.37 5.05
5.29 4.58 4.15 5.30 4.94
4.6
6.28 5.51 4.89 5.59 5.26 4.78
#NT: not tested